Recombinant Human Uncharacterized protein C17orf62 (C17orf62)

What is C17orf62 and what are its alternative names?

C17orf62 is now officially known as CYBC1 (Cytochrome b-245 chaperone 1) or EROS (Essential for Reactive Oxygen Species). It was initially identified as an uncharacterized open reading frame on chromosome 17 (C17orf62), but subsequent research revealed its crucial role in the phagocyte NADPH oxidase system . EROS was first discovered in 2017 through a mouse screen for host genetic factors that confer susceptibility to attenuated Salmonella typhimurium infection, where researchers found that an uncharacterized open reading frame bc017643 was highly conserved with the human ortholog C17ORF62 . Other alternative names include CGD5, referring to its association with chronic granulomatous disease . The gene encodes a protein that functions as a molecular chaperone for NOX2, a catalytic subunit of the phagocyte NADPH oxidase complex .

What is the primary function of CYBC1/EROS in cellular physiology?

CYBC1/EROS serves as a molecular chaperone that is essential for the maturation and transport of NOX2 through the Golgi apparatus . Its primary function is to regulate the abundance of the gp91phox-p22phox heterodimer, which constitutes the membrane-bound components of the phagocyte NADPH oxidase . EROS specifically associates with newly synthesized NOX2 polypeptide (58 kDa form) in the endoplasmic reticulum, protecting the catalytic subunit from rapid degradation . This binding precedes both heme incorporation into NOX2 and p22phox association with NOX2 . Through this chaperone activity, EROS enables proper assembly of the NADPH oxidase complex, which is crucial for the generation of reactive oxygen species during the respiratory burst—a critical antimicrobial defense mechanism in phagocytes . Studies in both genetically altered mice and transfected cell lines have confirmed this essential role in stabilizing the NOX2-p22phox heterodimer .

Where is CYBC1/EROS expressed in human tissues?

CYBC1/EROS appears to have a relatively broad tissue distribution. According to immunohistochemical staining of rat tissue cryosections, C17orf62 (CYBC1/EROS) was identified in all tissues examined, with antibodies consistently showing nuclear rim staining that confirmed its nuclear envelope residence across multiple tissue types . This contrasts with other nuclear envelope transmembrane proteins (NETs) that demonstrated more restricted tissue expression patterns in the same study . The protein is particularly important in cells of the immune system, especially those involved in phagocytosis and the respiratory burst, such as neutrophils and macrophages. This is consistent with its functional role in regulating the NADPH oxidase complex, which is most active in professional phagocytes . The protein has been studied in cell lines such as differentiated HL-60 cells (dHL-60), which serve as a model for neutrophil-like cells, further supporting its relevance in immune cell function .

How does CYBC1/EROS interact with other proteins in the NADPH oxidase complex?

CYBC1/EROS primarily interacts with the NOX2 (gp91phox) component of the NADPH oxidase complex, serving as a chaperone that stabilizes newly synthesized NOX2 in the endoplasmic reticulum . Colocalization studies using fluorescent confocal microscopy have demonstrated that EROS and NOX2 are expressed together in a scattered pattern throughout cells . This has been confirmed through multiple experimental approaches: 1) Immunofluorescence staining of differentiated HL-60 cells using FITC-labeled anti-EROS antibodies together with anti-NOX2 (7D5) antibodies and PE-labeled secondary antibodies showed overlapping expression patterns . 2) Fusion proteins of EROS–mRuby2 and Clover-NOX2 cotransfected into COS-7 cells further confirmed their colocalization . 3) NanoBiT-based protein interaction assays using N-LgBiT–EROS and NOX2–C-SmBit have been employed to study these interactions under various stimulation conditions . The binding of EROS to NOX2 occurs prior to heme incorporation and association with p22phox, suggesting that EROS plays an early role in the assembly pathway of the functional NADPH oxidase complex .

What are the most effective methods for detecting CYBC1/EROS expression in different experimental systems?

Several validated methods can be employed to detect CYBC1/EROS expression across different experimental systems:

• Western Blotting: Commercial monoclonal antibodies, such as the mouse monoclonal antibody clone OTI3B5, have been validated for Western blot applications with a recommended dilution of 1:4000 . This antibody has been validated using HEK293T cells transfected with pCMV6-ENTRY C17orf62 versus control plasmids, demonstrating specificity for the target protein .

• Immunofluorescence Microscopy: FITC-conjugated anti-EROS antibodies (e.g., CSB-PA859832LC01HU) have been successfully used for immunofluorescence staining of fixed cells . This approach can be combined with other markers, such as anti-NOX2 antibodies, to study colocalization patterns. Confocal microscopy of cells fixed with 4% paraformaldehyde, followed by antibody staining and DAPI counterstaining, has proven effective for visualizing EROS localization .

• Flow Cytometry: Flow cytometric analysis using FITC-conjugated anti-EROS antibodies has been successfully employed to detect EROS expression in differentiated HL-60 cells . This technique allows for quantitative assessment of expression levels and can be combined with surface markers to analyze specific cell populations.

• Fluorescent Fusion Proteins: Expression constructs encoding EROS fused to fluorescent proteins (e.g., EROS–C-mRuby2) can be transfected into various cell types for live-cell imaging of EROS localization and dynamics . This approach has been used successfully in COS-7 cells.

These methods can be adapted depending on the specific experimental question and system being studied. For optimal results, it is recommended to validate antibodies in your specific experimental system, as antibody performance can vary between applications and cell types.

How can researchers effectively produce recombinant CYBC1/EROS for structural and functional studies?

Production of high-quality recombinant CYBC1/EROS requires careful consideration of expression systems and purification strategies:

Expression System Selection:

Purification Strategy:

• Affinity Tags: Addition of affinity tags (His6, FLAG, GST) can facilitate purification. The tag position (N- or C-terminal) should be optimized to minimize interference with protein function.

• Membrane Protein Considerations: As EROS is predicted to be a membrane protein with two α-helices , detergent screening is crucial for extraction and maintaining stability during purification.

• Complex Formation: For structural studies of EROS in complex with NOX2-p22phox, co-expression strategies should be considered. The cryo-EM structure of the EROS–NOX2–p22phox–7G5 complex required careful optimization of complex formation and stability .

Quality Control:

• Size exclusion chromatography to assess homogeneity

• Functional assays to confirm activity (e.g., binding to NOX2)

• Mass spectrometry to verify protein identity and modifications

For researchers specifically interested in structural studies, the methods used to obtain the 3.56 Å resolution cryo-EM structure of the EROS–NOX2–p22phox–7G5 complex provide a valuable reference .

What are the known mutations of CYBC1/EROS associated with chronic granulomatous disease?

Mutations in CYBC1/EROS have been identified as a novel cause of chronic granulomatous disease (CGD), representing a newly recognized genetic basis for this primary immunodeficiency . While the full spectrum of disease-causing mutations continues to be characterized, the following key findings have been reported:

• Homozygous mutations that abolish EROS protein expression have been described in patients with CGD . These mutations result in severely reduced or absent production of reactive oxygen species by phagocytes, which is the hallmark of CGD.

• The first reported case involved a homozygous mutation that completely abolished EROS protein expression . This patient exhibited the typical clinical features of CGD, including increased susceptibility to bacterial and fungal infections.

• Since the initial discovery in 2018, several additional cases of this rare form of CGD secondary to CYBC1/EROS mutations have been reported . These cases have further confirmed the essential role of EROS in human immunity.

• Mechanistically, EROS mutations lead to deficiency of the gp91phox-p22phox heterodimer, which constitutes the membrane-bound components of the phagocyte NADPH oxidase . Without proper expression and stability of this heterodimer, phagocytes cannot generate the reactive oxygen species necessary for killing ingested microorganisms.

• The phenotype of EROS-deficient CGD is similar to that caused by mutations in the genes encoding the core components of the NADPH oxidase complex (gp91phox, p22phox, p47phox, p67phox, and p40phox) .

This relatively recent discovery highlights the importance of considering CYBC1/EROS mutations in patients with CGD-like phenotypes who lack mutations in the previously known causative genes.

How can researchers assess CYBC1/EROS functionality in cell-based assays?

Researchers can employ several approaches to assess CYBC1/EROS functionality in cell-based systems:

1. NADPH Oxidase Activity Assays:

• Isoluminol-Enhanced Chemiluminescence (ECL): This assay has been used to measure superoxide production in COS-91/22 cells (COS-7 cells expressing NOX2 and p22phox) . The assay can be performed in 96-well plate format, making it suitable for higher-throughput screening.

• Cytochrome c Reduction Assay: Measures superoxide production by monitoring the reduction of cytochrome c spectrophotometrically.

• Dihydrorhodamine 123 (DHR) Flow Cytometry: Can be used to assess intracellular ROS production in intact cells expressing EROS and NADPH oxidase components.

2. Protein Expression and Stability Analysis:

• Western Blotting: To measure the impact of EROS on NOX2 and p22phox protein levels in cells. This directly assesses EROS's chaperone function in stabilizing these proteins .

• Pulse-Chase Experiments: To determine the effect of EROS on the half-life of newly synthesized NOX2.

3. Protein-Protein Interaction Assays:

• NanoBiT-Based Assay: This approach has been successfully used to study EROS-NOX2 interactions in live cells . It involves cotransfection of cells with pcDNA3.1–N-LgBiT–EROS, pcDNA3.1–NOX2–C-SmBit, pcDNA3.1–p47phox, and pcDNA3.1–p67phox, followed by stimulation with PMA or buffer control and measurement of luminescence.

• Co-immunoprecipitation: To detect native protein complexes containing EROS and components of the NADPH oxidase.

• FRET or BiFC Assays: For visualizing protein interactions in live cells.

4. Subcellular Localization Studies:

• Confocal Microscopy: Using fluorescently labeled antibodies or fusion proteins to track EROS and NOX2 localization .

• Subcellular Fractionation: To biochemically determine the distribution of EROS in different cellular compartments.

5. Functional Complementation Assays:

• Rescue Experiments: In cells with CRISPR-mediated deletion of CYBC1/EROS, reintroduction of wild-type or mutant EROS can assess functional complementation.

6. FAD Binding Assays:

• Fluorescence Spectroscopy: To measure FAD binding to NOX2, which can be affected by EROS expression. The emission of FAD can be measured at 535 nm after excitation at 450 nm .

These assays provide complementary information about different aspects of EROS function and should be selected based on the specific research question.

What are the current challenges in studying CYBC1/EROS interactions with the NOX2-p22phox complex?

Despite significant progress in understanding CYBC1/EROS function, several challenges remain in studying its interactions with the NOX2-p22phox complex:

Structural Complexity:

• The membrane-associated nature of both EROS and the NOX2-p22phox complex presents technical challenges for structural and biochemical studies . While a high-resolution cryo-EM structure has been obtained, the dynamic nature of these interactions during biosynthesis and maturation remains difficult to capture.

• The precise timing and sequence of events involving EROS binding, heme incorporation, glycosylation, and p22phox association during NOX2 maturation require further elucidation .

Technical Limitations:

• Studying the transient interactions between EROS and newly synthesized NOX2 in the endoplasmic reticulum requires sophisticated approaches that can capture these fleeting interactions in their native environment.

• The proper reconstitution of functional NADPH oxidase components in heterologous expression systems often requires careful optimization of expression levels and conditions .

Functional Assessment:

• Distinguishing the direct effects of EROS on NOX2-p22phox stability from indirect effects on other aspects of NADPH oxidase assembly and function can be challenging.

• Developing assays that specifically measure EROS chaperone activity, separate from downstream effects on NADPH oxidase activity, remains difficult.

Physiological Regulation:

• The factors regulating EROS expression, localization, and activity in different cell types and under various physiological and pathological conditions are not fully understood.

• How EROS function is integrated with other chaperones and quality control mechanisms in the endoplasmic reticulum remains to be fully elucidated.

Disease-Associated Variants:

• The functional consequences of different EROS mutations found in CGD patients need more detailed characterization to understand structure-function relationships and potential therapeutic approaches .

Addressing these challenges will require integrated approaches combining structural biology, advanced imaging techniques, biochemical assays, and genetic models, preferably in physiologically relevant systems.

What are the optimal conditions for expressing recombinant CYBC1/EROS in heterologous systems?

Based on published methodologies, the following optimal conditions can be recommended for recombinant CYBC1/EROS expression:

Mammalian Expression Systems:

• Cell Lines: HEK293T cells have been successfully used for producing full-length human recombinant C17orf62/CYBC1/EROS . These cells provide appropriate post-translational modifications and folding environment for human membrane proteins.

• Expression Vectors: pcDNA3.1-based vectors have been effective for EROS expression in mammalian systems . For fusion proteins, constructs like EROS–C-mRuby2 have been successfully employed.

• Transfection Methods: Lipofectamine 3000 has been used effectively for transfection of EROS expression constructs into mammalian cells . Transfection should be performed according to manufacturer's protocols, typically with 24-hour expression periods.

• Co-expression Strategies: For functional studies involving NADPH oxidase components, co-transfection of EROS with NOX2, p22phox, p47phox, and p67phox has been successful . Optimizing the ratio of these plasmids may be necessary for proper complex formation.

Expression Optimization:

• Temperature: Standard mammalian cell culture conditions (37°C, 5% CO2) are typically used.

• Duration: Expression periods of 24-48 hours post-transfection have been reported .

• Induction Systems: For inducible expression, tetracycline-regulated systems can be considered to control expression levels.

Verification of Expression:

• Western Blotting: Anti-EROS antibodies with 1:4000 dilution have been validated for detecting expression .

• Fluorescence Microscopy: For fluorescent fusion proteins (e.g., EROS–C-mRuby2), direct visualization can confirm expression and localization .

Special Considerations:

• As a membrane protein with two predicted α-helices , EROS expression may benefit from optimization of membrane protein expression strategies, such as lower expression temperatures or specialized detergents for extraction.

• For structural studies requiring large quantities of protein, stable cell lines expressing EROS may be preferable to transient transfection.

By carefully optimizing these conditions, researchers can achieve reliable expression of functional CYBC1/EROS for various experimental applications.

How can researchers effectively perform co-immunoprecipitation studies with CYBC1/EROS?

Co-immunoprecipitation (Co-IP) is a valuable technique for studying protein-protein interactions involving CYBC1/EROS. Here's a methodological approach for effective Co-IP studies:

Reagent Selection:

• Antibodies: Select high-quality antibodies with validated specificity for CYBC1/EROS. Mouse monoclonal antibodies like clone OTI3B5 have been validated for various applications . For co-IP of interacting partners, antibodies against NOX2 (such as 7D5) can be used .

• Cell Lysis Buffers: Since EROS is predicted to be a membrane protein that interacts with membrane-bound components of NADPH oxidase, specialized lysis conditions are required:

  • For membrane proteins, use buffers containing mild detergents like digitonin (0.5-1%), CHAPS (0.5-1%), or n-dodecyl-β-D-maltoside (0.5-1%)

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying phosphorylation-dependent interactions

Protocol Overview:

• Cell Preparation:

  • Use cells expressing endogenous proteins (e.g., differentiated HL-60 cells) or transfected cells (e.g., HEK293T cells transfected with EROS and interaction partners)

  • Harvest 1-2 × 10^7 cells per condition

  • Wash cells with ice-cold PBS

• Cell Lysis:

  • Lyse cells in 1 ml of appropriate lysis buffer for 30 minutes on ice with occasional mixing

  • Centrifuge at 14,000 × g for 10 minutes at 4°C to remove insoluble material

  • Transfer supernatant to a new tube and determine protein concentration

• Pre-clearing (Optional):

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add 2-5 μg of primary antibody to 500 μg of pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add 50 μl of Protein A/G beads and incubate for 2-4 hours at 4°C

  • Wash beads 4-5 times with lysis buffer

  • Elute proteins with SDS sample buffer by heating at 95°C for 5 minutes

• Analysis:

  • Separate proteins by SDS-PAGE

  • Perform western blotting using antibodies against EROS and potential interacting partners (NOX2, p22phox)

Controls and Validations:

• Negative Controls: Use non-immune IgG or lysates from cells not expressing the target protein

• Input Control: Analyze a small portion of the lysate before immunoprecipitation

• Reciprocal Co-IP: Confirm interactions by performing the IP in reverse (using antibody against the interacting partner)

• Competition Assays: Include excess recombinant protein to compete for binding

This approach should enable researchers to effectively study the interactions between CYBC1/EROS and components of the NADPH oxidase complex or other potential binding partners.

What are the recommended approaches for studying CYBC1/EROS localization in cells?

Multiple complementary approaches can be employed to study CYBC1/EROS localization in cells, each with distinct advantages:

1. Fluorescent Confocal Microscopy:

• Fixed Cell Immunofluorescence: This approach has been successfully used to visualize EROS localization in differentiated HL-60 cells . The protocol involves:

  • Fixing cells with 4% paraformaldehyde

  • Incubating with anti-EROS–FITC antibodies overnight at 4°C

  • For co-localization studies, including additional primary antibodies (e.g., anti-NOX2 7D5)

  • Adding appropriate secondary antibodies (e.g., goat anti-mouse PE)

  • Counterstaining nuclei with DAPI

  • Imaging using a confocal microscope such as LEICA TCS SP8

• Live-Cell Imaging with Fluorescent Fusion Proteins: This method allows real-time visualization of protein dynamics:

  • Transfect cells with constructs encoding EROS fused to fluorescent proteins (e.g., EROS–C-mRuby2)

  • For co-localization, co-transfect with fusion proteins of interaction partners (e.g., Clover-NOX2)

  • Culture transfected cells on glass coverslips

  • Mount coverslips in chambers suitable for live-cell imaging

  • Image using a confocal microscope with appropriate filters

2. Subcellular Fractionation and Biochemical Analysis:

• Separate cellular components (cytosol, membrane, nuclear, and organelle fractions)

• Analyze fractions by western blotting using anti-EROS antibodies

• Include markers for different cellular compartments (e.g., calnexin for ER, GM130 for Golgi)

• This approach complements imaging by providing quantitative distribution data

3. Immuno-Electron Microscopy:

• For ultrastructural localization at nanometer resolution

• Particularly valuable for precise localization within membrane structures

• Can distinguish between different ER subdomains or Golgi compartments

4. Proximity Labeling Techniques:

• BioID or APEX2 fusion proteins to identify the proximal proteome of EROS

• These approaches can reveal the subcellular neighborhood of EROS

5. Correlative Light and Electron Microscopy (CLEM):

• Combines fluorescence microscopy with electron microscopy

• Enables visualization of EROS at both cellular and ultrastructural levels

6. Flow Cytometry:

• For quantitative assessment of EROS expression in cell populations

• Particularly useful for analyzing expression in primary cells or heterogeneous populations

• Has been successfully applied to study EROS expression in differentiated HL-60 cells

Software for Analysis:

• For colocalization analysis, software packages like ImageJ with the JACoP plugin or Imaris can quantify the degree of overlap between EROS and other proteins of interest.

These approaches should be selected based on the specific research question, with multiple techniques often providing complementary information about EROS localization and dynamics.

How can CRISPR-Cas9 be utilized to study CYBC1/EROS function?

CRISPR-Cas9 genome editing provides powerful approaches for studying CYBC1/EROS function through various strategies:

1. Gene Knockout Studies:

• Complete Gene Deletion: CRISPR-mediated deletion of CYBC1/EROS has been successfully performed to study its function in human cells . This approach can reveal the consequences of complete loss of EROS expression.

• Design Considerations:

  • Target early exons to ensure complete disruption of protein function

  • Design multiple guide RNAs to increase efficiency

  • Screen edited clones by genomic PCR, sequencing, and western blotting to confirm knockout

2. Knock-in of Reporter Tags:

• Endogenous Tagging: Insert fluorescent proteins (GFP, mCherry) or epitope tags (FLAG, HA) at the C-terminus of endogenous CYBC1/EROS

• Benefits:

  • Visualize endogenous protein localization

  • Enable pull-down of protein complexes without overexpression artifacts

  • Monitor endogenous protein dynamics

3. Introduction of Disease-Associated Mutations:

• Precision Editing: Generate specific point mutations identified in CGD patients

• Homology-Directed Repair (HDR): Use donor templates containing the desired mutation

• Applications:

  • Study structure-function relationships

  • Recreate patient-specific mutations to understand disease mechanisms

  • Test potential therapeutic approaches

4. Conditional/Inducible Systems:

• Floxed Alleles: Generate conditional knockout cells by inserting loxP sites flanking critical exons

• Tet-On/Off Systems: Combine CRISPR with tetracycline-inducible expression

• Benefits:

  • Study acute vs. chronic loss of EROS

  • Avoid compensatory mechanisms that may develop in constitutive knockouts

  • Enable tissue-specific or temporal control of expression

5. CRISPR Interference/Activation (CRISPRi/CRISPRa):

• Reversible Modulation: Use deactivated Cas9 (dCas9) fused to transcriptional repressors (CRISPRi) or activators (CRISPRa)

• Applications:

  • Tune CYBC1/EROS expression levels without permanent genetic changes

  • Study dose-dependent effects

  • Examine consequences of overexpression

6. Screening Approaches:

• CRISPR Libraries: Screen for genes that interact with CYBC1/EROS

• Phenotypic Readouts:

  • NADPH oxidase activity (e.g., ROS production)

  • NOX2 protein expression levels

  • Cell survival under oxidative stress conditions

7. Base or Prime Editing:

• Precise Modification: For introducing specific mutations without DNA breaks

• Applications:

  • Generate precise disease-relevant mutations

  • Study regulatory elements affecting CYBC1/EROS expression

When implementing these approaches, careful validation of editing efficiency and specificity is essential, including sequencing confirmation and analysis of potential off-target effects.

What are the best methods for assessing NADPH oxidase activity in relation to CYBC1/EROS?

Given CYBC1/EROS's critical role in regulating the phagocyte NADPH oxidase, several complementary assays can be used to assess oxidase activity in relation to EROS function:

1. Cell-Free Assays:

• Isoluminol-Enhanced Chemiluminescence (ECL): This highly sensitive assay has been successfully used to measure superoxide production in reconstituted systems . The protocol involves:

  • Preparing membrane fractions from cells expressing various components

  • Setting up reactions in 96-well white tissue culture plates

  • Adding isoluminol and peroxidase as detection reagents

  • Initiating the reaction with activators (e.g., PMA)

  • Measuring luminescence using a plate reader over time

• Cytochrome c Reduction Assay:

  • Measures superoxide production based on the reduction of cytochrome c

  • Quantified spectrophotometrically at 550 nm

  • Can be performed with intact cells or subcellular fractions

2. Cellular Assays:

• Dihydrorhodamine 123 (DHR) Flow Cytometry:

  • Incubate cells with DHR-123, which is oxidized to fluorescent rhodamine 123 by ROS

  • Stimulate cells with activators like PMA or opsonized particles

  • Analyze by flow cytometry to quantify the fluorescence intensity

  • Particularly useful for analyzing primary cells or mixed populations

• Nitroblue Tetrazolium (NBT) Reduction:

  • Simple colorimetric assay where NBT is reduced to formazan (blue precipitate)

  • Can be quantified by microscopy (% positive cells) or spectrophotometry

  • Commonly used for diagnostic testing of CGD

• Amplex Red Assay:

  • Detects H2O2 (the dismutation product of superoxide)

  • Higher sensitivity than cytochrome c reduction

  • Less interference from cellular components

3. Advanced Imaging Approaches:

• Live-Cell Imaging with ROS-Sensitive Probes:

  • Genetically encoded probes (HyPer, roGFP)

  • Chemical probes (CellROX, H2DCFDA)

  • Enables real-time visualization of ROS production with subcellular resolution

• Single-Cell Analysis:

  • Microfluidic systems for measuring ROS from individual cells

  • Correlating ROS production with other cellular parameters

4. Molecular Assays:

• FAD Binding Assessment:

  • Measure FAD incorporation into NOX2, which is influenced by EROS

  • FAD emission can be measured at 535 nm after excitation at 450 nm using fluorescence spectroscopy

• NOX2 Maturation Analysis:

  • Monitor glycosylation state of NOX2 by western blot

  • Track the conversion of the 58 kDa precursor to the fully glycosylated 91 kDa form

5. Functional Readouts in Whole Cells:

• Microbial Killing Assays:

  • Quantify killing of microorganisms (e.g., S. aureus, Candida)

  • Provides a physiologically relevant measure of NADPH oxidase function

• NETosis Measurement:

  • Assess formation of neutrophil extracellular traps, which depends on ROS

  • Can be quantified by DNA release or microscopy

When designing experiments, researchers should consider including both positive controls (cells with known NADPH oxidase activity) and negative controls (cells lacking critical components of the oxidase, such as those from CGD patients). Additionally, specific inhibitors of NADPH oxidase (e.g., diphenyleneiodonium) can help confirm the specificity of the detected signals.